Elsevier

Neurobiology of Disease

Volume 85, January 2016, Pages 93-98
Neurobiology of Disease

Striatal NELF-mediated RNA polymerase II stalling controls l-dopa induced dyskinesia

https://doi.org/10.1016/j.nbd.2015.10.013Get rights and content

Highlights

  • L-3,4-dihydroxyphenylalanine (L-Dopa)-induced dyskinesia are related to an overexpression of immediate-early genes (IEG).

  • IEG rapid transcription involves the stalling of RNA polymerase II on IEG promoters.

  • Negative elongation factor (NELF) protein complex controls stalling.

  • Repression of NELF-mediating RNA polymerase II stalling achieves antidyskinetic effect.

  • These results highlight the role of transcriptional events in dyskinesia establishment, acute dyskinetic manifestation and in the therapeutic response to L-Dopa.

Abstract

Long-term l-3,4-dihydroxyphenylalanine (l-Dopa) treatment in Parkinson's disease leads to involuntary movements called dyskinesia, notably through an overexpression of immediate-early genes (IEG). Their rapid transcription involves the stalling of RNA polymerase II on IEG promoters, a mechanism that critically depends on the presence of the negative elongation factor (NELF) protein complex. We here down-regulated the key NELF-E subunit using lentiviral vector delivery of a short hairpin RNA in the striatum of 6-hydroxydopamine lesioned rats. Such NELF-E reduced expression significantly attenuated the development of abnormal involuntary movements in response to chronic l-Dopa treatment. Effectiveness of silencing was demonstrated by the significant decrease in striatal ∆ FosB, ARC and Zif268 IEG expression. Repression of NELF-mediating RNA polymerase II stalling thus achieves both antidyskinetic and potentiation of antiparkinsonian l-Dopa effect, highlighting the role of transcriptional events in dyskinesia establishment, acute dyskinetic manifestation and in the therapeutic response to l-Dopa.

Introduction

The most effective symptomatic therapy in Parkinson's disease (PD) remains the dopamine precursor l-3,4-dihydroxyphenylalanine (l-Dopa). Long-term treatment leads to involuntary aimless movements called l-Dopa-induced dyskinesia (LID) (Bastide et al., 2015, Fahn, 2008, Stocchi et al., 1997), which first causative event is a l-Dopa-induced striatal overexpression of several molecular markers, in particular the members of the immediate-early gene (IEG) family (Bastide et al., 2015), a class of genes rapidly transcribed in response to an external stimulus, including ∆ FosB, ARC and Zif268 (Bastide et al., 2014, Berke et al., 1998, Gerfen et al., 1990, Gerfen et al., 1995, McClung et al., 2004). Down-regulating expression of ∆ FosB for instance, decreases LID severity both in rodent (Andersson et al., 1999) and non-human primates (Berton et al., 2009). While the mechanisms of rapid IEG transcription remain unclear, recent evidences suggest that expression of many IEGs depends on a prior recruitment of the RNA polymerase II, which initiates transcription elongation and stalls after transcribing a short piece of mRNA near the promoter (Lis, 1998, Nechaev and Adelman, 2011, Saha and Dudek, 2013, Saha et al., 2011). RNA polymerase II stalling is critically regulated by a protein complex, the negative elongation factor (NELF), composed of four essential subunits: NELF-A, -B, -C/D and -E (Narita et al., 2003, Saha and Dudek, 2013, Saha et al., 2011). NELF-mediated RNA polymerase II stalling on IEG promoters poises them for rapid transcription within few minutes after an external stimulus (Saha et al., 2011). While the in vitro machinery is well described, the role of NELF-mediated RNA polymerase II stalling remains however to be demonstrated in vivo in physiological and pathological states.

Contrary to what the name of the mechanism (i.e. stalling) suggests, reducing stalling should actually leads to a reduction in rapid transcription of IEGs. We thus hypothesize that such functional inhibition of rapid IEG transcription would lead to reduction of IEG expression and to less severe LID. Therefore, to assess the precise role of NELF-mediated RNA polymerase II stalling upon LID severity, we down-regulated the NELF-E subunit expression by RNA interference (RNAi), using a lentiviral (LV) vector delivering a short hairpin RNA (shRNA) in the striatum of dyskinetic 6-hydroxydopamine (6-OHDA) lesioned rats and we quantified the l-Dopa induced abnormal involuntary movements (AIMs), the rodent analog of LID. We then quantified the striatal expression of ∆ FosB, ARC and Zif268 to assess the impact of NELF-mediated RNA polymerase II stalling on IEG expression.

Section snippets

Design of LV vectors and NELF-E reduced expression validation

shRNA LV plasmids (pLKO.1) (Dehay et al., 2012) carrying pre-designed short hairpins sequences for NELF-E or scrambled hairpin sequences were purchased from Sigma-Aldrich (USA). The shRNA sequence used to target NELF-E mRNA is: CCG GCT GGA TTC CTT GTG CCT CAT ACT CGA GTA TGA GGC ACA AGG AAT CCA GTT TTT G (TRC Number: TRCN0000074958) (Saha et al., 2011). Lentiviral production (final titer of 2.55 × 108 pI/mL) was performed in the IFR 66 vectorology platform (University of Bordeaux, France) by

Results

We first explored the level of NELF-E reduced expression in rat striatal primary cell cultures infected by both LV shRNA NELF-E (n = 3) and scramble (n = 3) as previously described in details (Berthet et al., 2012, Dehay et al., 2012, Martin-Negrier et al., 2006). LV shRNA NELF-E induced a 47% decrease in NELF-E protein levels (p < 0.05) (Fig. 1A). Such in vitro evidence was followed by the in vivo demonstration that the shRNA NELF-E is effective in reducing the expression of NELF-E in transduced

Discussion

l-Dopa, the mainstay treatment for PD, rapidly induces fluctuations and LID. A rational approach from the prospect of novel pharmacotherapies should first identify and then exploit the biology that separates LID from the l-DOPA-induced therapeutic effects. LIDs are associated with both presynaptic and postsynaptic striatal mechanisms (Bezard et al., 2001, Jenner, 2008), including an impressive and rapid overexpression of ∆ FosB, ARC and Zif268 IEGs (Bastide et al., 2014, Berke et al., 1998,

Author contributions

M.B., S.B. and N.D. conducted the experiments, E.B. designed the experiments, M.B., S.B., N.D. and E.B. wrote the paper.

Conflict of interest

The authors declare having no conflict of interest.

Acknowledgments

This work was supported by Agence Nationale de la Recherche (EB: ANR-07-MNP-Trafinlid), the Fondation de France (EB) and grant LABEX BRAINANR-10-LABX-43 (EB). MB is the recipient of an MESR grant. The Université Bordeaux Segalen and the Centre National de la Recherche Scientifique provided infrastructural support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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